Novel methods for the stabilisation of immunoglobulin constant domains

Abstract

The present invention relates to a method of producing a modified protein having an increased stability and/or improved folding efficiency as compared to the unmodified protein, the method comprising (i) modifying a nucleic acid molecule encoding a protein comprising at least one immunoglobulin constant domain-like region by (a-i) replacing the nucleotides encoding at least one amino acid, preferably an uncharged amino acid, in the loop separating the C strand from the D strand with nucleotides encoding a charged amino acid selected from the group consisting of Arg, Lys, His, Glu and Asp and/or replacing the nucleotides encoding at least one amino acid, preferably an uncharged amino acid, in the helix connecting the E strand with the F strand with nucleotides encoding a charged amino acid selected from the group consisting of Arg, Lys, His, Glu and Asp; (a-ii) replacing the nucleotides encoding at least one amino acid not having a side chain that can form a hydrogen bond in the loop separating the C strand from the D strand with nucleotides encoding an amino acid having a side chain capable of forming a hydrogen bond selected from the group consisting of Gln, Asn, Tyr, Ser and Thr and/or replacing the nucleotides encoding at least one amino acid not having a side chain that can form a hydrogen bond in the helix connecting the E strand with the F strand with nucleotides encoding an amino acid having a side chain capable of forming a hydrogen bond selected from the group consisting of Gln, Asn, Tyr, Ser and Thr; and/or (a-iii) replacing the nucleotides encoding at least one amino acid in the loop separating the C strand from the D strand with nucleotides encoding a cysteine and/or replacing the nucleotides encoding at least one amino acid in the helix connecting the E strand with the F strand with nucleotides encoding a cysteine; and/or (b) replacing the nucleotides encoding at least one non-hydrophobic amino acid at a position suitable to participate in the formation of the hydrophobic core with nucleotides encoding a hydrophobic amino acid selected from the group consisting of Val, Ile, Leu, Met, Phe, Trp and Pro; and (ii) expressing the nucleic acid molecule to produce the stabilised protein. The present invention further relates to a method of producing a modified protein having an improved secretion from cells as compared to the unmodified protein, as well as to a protein comprising at least one immunoglobulin constant domain-like region having an additional salt bridge, an additional hydrogen bond, an additional disulfide bridge and/or an extended hydrophobic core. The present invention further relates to a nucleic acid molecule encoding the modified protein of the invention, as well as a vector comprising said nucleic acid molecule and a host cell comprising the vector. Further, the present invention relates also to a composition as well as to a kit.

1 . A method of producing a modified protein having an increased stability and/or improved folding efficiency as compared to the unmodified protein, the method comprising
 (i) modifying a nucleic acid molecule encoding a protein comprising at least one immunoglobulin constant domain-like region by
 (a-i) replacing the nucleotides encoding at least one amino acid, preferably an uncharged amino acid, in the loop separating the C strand from the D strand with nucleotides encoding a charged amino acid selected from the group consisting of Arg, Lys, His, Glu and Asp and/or replacing the nucleotides encoding at least one amino acid, preferably an uncharged amino acid, in the helix connecting the E strand with the F strand with nucleotides encoding a charged amino acid selected from the group consisting of Arg, Lys, His, Glu and Asp; 
 (a-ii) replacing the nucleotides encoding at least one amino acid not having a side chain that can form a hydrogen bond in the loop separating the C strand from the D strand with nucleotides encoding an amino acid having a side chain capable of forming a hydrogen bond selected from the group consisting of Gln, Asn, Tyr, Ser and Thr and/or replacing the nucleotides encoding at least one amino acid not having a side chain that can form a hydrogen bond in the helix connecting the E strand with the F strand with nucleotides encoding an amino acid having a side chain capable of forming a hydrogen bond selected from the group consisting of Gln, Asn, Tyr, Ser and Thr; and/or 
 (a-iii) replacing the nucleotides encoding at least one amino acid in the loop separating the C strand from the D strand with nucleotides encoding a cysteine and/or replacing the nucleotides encoding at least one amino acid in the helix connecting the E strand with the F strand with nucleotides encoding a cysteine; and/or 
 (b) replacing the nucleotides encoding at least one non-hydrophobic amino acid at a position suitable to participate in the formation of the hydrophobic core with nucleotides encoding a hydrophobic amino acid selected from the group consisting of Val, Ile, Leu, Met, Phe, Trp and Pro; and 
   (ii) expressing the nucleic acid molecule to produce the stabilised protein.   
     
     
         2 . A method of producing a modified protein having an improved secretion from cells as compared to the unmodified protein, the method comprising
 (i) modifying a nucleic acid molecule encoding a protein comprising at least one immunoglobulin constant domain-like region by
 (a-i) replacing the nucleotides encoding at least one amino acid, preferably an uncharged amino acid, in the loop separating the C strand from the D strand with nucleotides encoding a charged amino acid selected from the group consisting of Arg, Lys, His, Glu and Asp and/or replacing the nucleotides encoding at least one amino acid, preferably an uncharged amino acid, in the helix connecting the E strand with the F strand with nucleotides encoding a charged amino acid selected from the group consisting of Arg, Lys, His, Glu and Asp; 
 (a-ii) replacing the nucleotides encoding at least one amino acid not having a side chain that can form a hydrogen bond in the loop separating the C strand from the D strand with nucleotides encoding an amino acid having a side chain capable of forming a hydrogen bond selected from the group consisting of Gln, Asn, Tyr, Ser and Thr and/or replacing the nucleotides encoding at least one amino acid not having a side chain that can form a hydrogen bond in the helix connecting the E strand with the F strand with nucleotides encoding an amino acid having a side chain capable of forming a hydrogen bond selected from the group consisting of Gln, Asn, Tyr, Ser and Thr; and/or 
 (a-iii) replacing the nucleotides encoding at least one amino acid in the loop separating the C strand from the D strand with nucleotides encoding a cysteine and/or replacing the nucleotides encoding at least one amino acid in the helix connecting the E strand with the F strand with nucleotides encoding a cysteine; and/or 
 (b) replacing the nucleotides encoding at least one non-hydrophobic amino acid at a position suitable to participate in the formation of the hydrophobic core with nucleotides encoding a hydrophobic amino acid selected from the group consisting of Val, Ile, Leu, Met, Phe, Trp and Pro; and 
   (ii) expressing the nucleic acid molecule to produce the protein having improved secretion.   
     
     
         3 . A protein comprising at least one immunoglobulin constant domain-like region having an additional salt bridge, an additional hydrogen bond, an additional disulfide bond and/or an extended hydrophobic core, wherein the protein comprises one or more modifications selected from the group consisting of
 (i) a replacement of at least one amino acid, preferably an uncharged amino acid, in the loop separating the C strand from the D strand for a charged amino acid selected from the group consisting of Arg, Lys, His, Glu and Asp and/or a replacement of at least one amino acid, preferably an uncharged amino acid, in the helix connecting the E strand with the F strand for a charged amino acid selected from the group consisting of Arg, Lys, His, Glu and Asp, thereby enabling the formation of an additional salt bridge;   (ii) a replacement of at least one amino acid not having a side chain that can form a hydrogen bond in the loop separating the C strand from the D strand for an amino acid having a side chain capable of forming a hydrogen bond selected from the group consisting of Gln, Asn, Tyr, Ser and Thr and/or a replacement of at least one amino acid not having a side chain that can form a hydrogen bond in the helix connecting the E strand with the F strand for an amino acid having a side chain capable of forming a hydrogen bond selected from the group consisting of Gln, Asn, Tyr, Ser and Thr; thereby enabling the formation of an additional hydrogen bond;   (iii) a replacement of at least one amino acid in the loop separating the C strand from the D strand for a cysteine and/or a replacement of at least one amino acid in the helix connecting the E strand with the F strand for a cysteine; thereby enabling the formation of an additional disulfide bridge; and/or   (iv) a replacement of at least one non-hydrophobic amino acid at a position suitable to participate in the formation of the hydrophobic core for a hydrophobic amino acid selected from the group consisting of Val, Ile, Leu, Met, Phe, Trp and Pro;   thereby extending the hydrophobic core.   
     
     
         4 . The method according to  claim 1  or  2  or the protein of  claim 3 , wherein the at least one amino acid in the loop separating the C strand from the D strand is an amino acid at a position corresponding to position 48 in SEQ ID NO: 1 and wherein the at least one amino acid in the helix connecting the E strand with the F strand is an amino acid at a position corresponding to position 76 in SEQ ID NO: 1. 
     
     
         5 . The method according to any one of  claim 1 ,  2  or  4  or the protein according to  claim 3  or  4 , wherein the at least one non-hydrophobic amino acid in a position suitable to participate in the formation of the hydrophobic core is an amino acid in the D strand. 
     
     
         6 . The method according to  claim 5  or the protein according to  claim 5 , wherein the at least one non-hydrophobic amino acid in the D strand is an amino acid at a position corresponding to position 52 in SEQ ID NO: 1. 
     
     
         7 . The method or the protein according to any one of the preceding claims, wherein the protein comprising at least one immunoglobulin constant domain-like region is an antibody. 
     
     
         8 . The method or the protein according to  claim 7 , wherein the antibody is a human or humanised antibody. 
     
     
         9 . The method or the protein according to any one of the preceding claims, wherein the protein comprising at least one immunoglobulin constant domain-like region is a eukaryotic protein. 
     
     
         10 . A nucleic acid molecule encoding the protein obtained by the method of any one of  claims 1 ,  2  or  4  to  9  or the protein of any one of  claims 3  to  9 . 
     
     
         11 . A vector comprising the nucleic acid molecule of  claim 10 . 
     
     
         12 . A host cell or a non-human host transformed with the vector of  claim 11 . 
     
     
         13 . A method for the production of a modified protein according to  claim 3 , the method comprising the steps of:
 (i) culturing the host cell of  claim 12  under suitable conditions; and   (ii) isolating the modified protein according to  claim 3  produced by said host cell.   
     
     
         14 . A composition comprising at least one of
 (i) the protein of any one of  claims 3  to  9 ;   (ji) the nucleic acid molecule of  claim 10 ;   (iii) the vector of  claim 11 ; or   (iv) the host cell of  claim 12 .   
     
     
         15 . A kit comprising at least one of
 (i) the protein of any one of  claims 3  to  9 ;   (ji) the nucleic acid molecule of  claim 10 ;   (iii) the vector of  claim 11 ; or   (iv) the host cell of  claim 12 .